Hypertension is a major risk factor for the development of cardiovascular disease, i.e., stroke heart failure and peripheral vascular disease. Arteries from animals with genetic and mineralocorticoid-NaCl hypertension are known to exhibit increases in steady-state Cl- turnover and vasoconstrictor responsiveness. The relationship between Cl- and vascular contraction has not been clearly defined. The proposed research program focuses on the cellular/molecular mechanisms underlying altered Cl- permeability and its contribution to increased vascular reactivity in hypertension. The overall plan is designed to probe the physiological role of specific Cl- transporters/channels in modulating transmembrane signaling events (Ca2+, pHi, membrane potential) involved in contractile agonist stimulation of vascular smooth muscle cells (VSMC) isolated from arteries (aorta, caudal artery) of normotensive, spontaneously hypertensive (NaCl- sensitive and resistant strains), and aldosterone-NaCl hypertension. Combination of fluorescence microscopy, newly-developed digital imaging methods, and patch-clamp techniques will be used to probe the molecular mechanisms of VSMC Cl- homeostasis. The specific aims are: 1) Define the cellular pathways involved in Cl- homeostasis in cultured, as well as freshly isolated VSMC. Ion-selective fluorescent dyes, fluorescence microcopy, and patch-clamp techniques will be used to analyze in detail different Cl- transporters/channels in intact, single VSMC; 2) Determine the molecular mechanisms involved in Cl- transporter/channel activation. Fluorescence microscopy in combination with digital image analysis methods and patch-clamp techniques will be used to make simultaneous spatial/temporal measurements of Cl- conductances and different ions following agonists stimulation; 3) Probe the cellular/molecular mechanisms underlying increased VSMC Cl- permeability and its contribution to increase VSMC reactivity. Furthermore, we will evaluate the contribution of increased Cl- permeability to enhanced vascular responsiveness in aorta and caudal artery using different Cl- flux inhibitors. These studies will contribute to our basic understanding of the cellular/molecular mechanisms underlying normal and altered VSMC ion permeability in relationship to VSMC dysfunction in the pathogenesis of hypertension, atherosclerosis and arterial vasospasm.